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Duc, Nguyen Minh,Du, Yang,Thorsen, Thor S.,Lee, Su Youn,Zhang, Cheng,Kato, Hideaki,Kobilka, Brian K.,Chung, Ka Young Springer New York LLC 2015 Journal of the American Society for Mass Spectrome Vol.26 No.5
<P>G protein-coupled receptors (GPCRs) have important roles in physiology and pathology, and 40% of drugs currently on the market target GPCRs for the treatment of various diseases. Because of their therapeutic importance, the structural mechanism of GPCR signaling is of great interest in the field of drug discovery. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for analyzing ligand binding sites, the protein-protein interaction interface, and conformational changes of proteins. However, its application to GPCRs has been limited for various reasons, including the hydrophobic nature of GPCRs and the use of detergents in their preparation. In the present study, we tested the application of bicelles as a means of solubilizing GPCRs for HDX-MS studies. GPCRs (e.g., β<SUB>2</SUB>-adrenergic receptor [β<SUB>2</SUB>AR], μ-opioid receptor, and protease-activated receptor 1) solubilized in bicelles produced better sequence coverage (greater than 90%) than GPCRs solubilized in n-dodecyl-β-D-maltopyranoside (DDM), suggesting that bicelles are a more effective method of solubilization for HDX-MS studies. The HDX-MS profile of β<SUB>2</SUB>AR in bicelles showed that transmembrane domains (TMs) undergo lower deuterium uptake than intracellular or extracellular regions, which is consistent with the fact that the TMs are highly ordered and embedded in bicelles. The overall HDX-MS profiles of β<SUB>2</SUB>AR solubilized in bicelles and in DDM were similar except for intracellular loop 3. Interestingly, we detected EX1 kinetics, an important phenomenon in protein dynamics, at the C-terminus of TM6 in β<SUB>2</SUB>AR. In conclusion, we suggest the application of bicelles as a useful method for solubilizing GPCRs for conformational analysis by HDX-MS.</P> [FIG OMISSION]</BR>
Solid-state NMR Studies of Membrane Proteins Using Phospholipid Bicelles
Kim, Yong-Ae Korean Chemical Society 2006 Bulletin of the Korean Chemical Society Vol.27 No.3
Membrane proteins in highly oriented lipid bilayer samples are useful for membrane protein structure determination. We used in the past planar lipid bilayers which were aligned and supported on the glass slide. These samples were mechanically aligned in a magnetic field. However, these stacks of glass slides with planar lipid bilayers are not well suited for use with a commercial solid-state NMR probe with a round coil. Therefore, a homebuilt solid-state NMR probe was built and used with a stack of thin glass plates wherein the RF coil was wrapped directly around the flat square sample. Recently, we began to use magnetically aligned bicelles that are suitable for the structure determination of membrane proteins by solid-state NMR spectroscopy without any effort to build a flat square coil probe. These bicelle samples are well suited for use with a commercial solidstate NMR probe with a round coil, are very easy to prepare and are very stable, so that they can be kept for more than a year. In this paper, we present the solid-state NMR spectra of optimized and magnetically oriented bicelle samples of membrane proteins.
Park, Tae-Joon,Kim, Ji-Sun,Um, Seung-Hoon,Kim, Yong-Ae Korean Chemical Society 2010 Bulletin of the Korean Chemical Society Vol.31 No.5
$^1H-^{15}N$ heteronuclear dipolar coupling solid-state NMR experiments on lipid bilayer or bicelle samples are very useful for the structural studies of membrane proteins. However, to study these biological samples using solid-state NMR, a specific probe with high efficiency and high capability is required. In this paper, we describe the optimized design, construction, and efficiency of a 400 MHz wide-bore $^1H-^{15}N$ solid-state NMR probe with 5-mm solenoidal rf coil for high power, multi-pulse sequence experiments, such as 2D PISEMA or 2D SAMMY.
Park, Tae-Joon,Choi, Sung-Sub,Jung, Ji-Ho,Park, Yu-Geun,Kim, Yongae Korean Chemical Society 2013 Bulletin of the Korean Chemical Society Vol.34 No.3
A low-${\varepsilon}$ solid-state NMR(Nuclear Magnetic Resonance) probe was developed for the spectroscopic analysis of two-dimensional $^{15}N-^1H$ heteronuclear dipolar coupling in dilute membrane proteins oriented in hydrated and dielectrically lossy lipid environments. The system employed a 800 MHz narrow-bore magnet. A solenoid coil strip shield was used to reduce deleterious RF sample heating by minimizing the conservative electric fields generated by the double-tuned resonator at high magnetic fields. The probe's design, construction, and performance in solid-state NMR experiments at high magnetic fields are described here. Such high-resolution solid-state NMR spectroscopic analysis of static oriented samples in hydrated phospholipid bilayers or bicelles could aid the structural analysis of dilute biological membrane proteins.
Tae-Joon Park,김지선,Seung-Hoon Um,김용애 대한화학회 2010 Bulletin of the Korean Chemical Society Vol.31 No.5
1H-15N heteronuclear dipolar coupling solid-state NMR experiments on lipid bilayer or bicelle samples are very useful for the structural studies of membrane proteins. However, to study these biological samples using solid-state NMR, a specific probe with high efficiency and high capability is required. In this paper, we describe the optimized design, construction,and efficiency of a 400 MHz wide-bore 1H-15N solid-state NMR probe with 5-mm solenoidal rf coil for high power, multi-pulse sequence experiments, such as 2D PISEMA or 2D SAMMY.
박태준,최성섭,정지호,Yu-Geun Park,김용애 대한화학회 2013 Bulletin of the Korean Chemical Society Vol.34 No.3
A low-ε solid-state NMR(Nuclear Magnetic Resonance) probe was developed for the spectroscopic analysis of two-dimensional 15N-1H heteronuclear dipolar coupling in dilute membrane proteins oriented in hydrated and dielectrically lossy lipid environments. The system employed a 800 MHz narrow-bore magnet. A solenoid coil strip shield was used to reduce deleterious RF sample heating by minimizing the conservative electric fields generated by the double-tuned resonator at high magnetic fields. The probe's design, construction, and performance in solid-state NMR experiments at high magnetic fields are described here. Such high-resolution solid-state NMR spectroscopic analysis of static oriented samples in hydrated phospholipid bilayers or bicelles could aid the structural analysis of dilute biological membrane proteins.
Bong Su Kang,Sunghak Choi,Shogo Taguchi,Keishi Suga,Hiroshi Umakoshi,Keesung Kim,Moon Kyu Kwak,Hosup Jung 한국정밀공학회 2024 International Journal of Precision Engineering and Vol.11 No.2
Although a microfluidic technique for vesicle synthesis has drawn attention in the biomedical field due to its superiority in size control and monodispersity, it has suffered from the extraction of residual solvent. Previous studies attempted the solvent-free microfluidic method using the bicelle-to-vesicle transition, but only a limited size of vesicles was obtained with low membrane properties on account of inefficient mixing. In this paper, we suggest the solvent-free and non-stimulus method for the vesicle preparation using a microfluidic chip with different types of mixing structures, among which the tilted-type structure performed an efficient mixing. With this micromixer, lipid vesicles can be continuously obtained at high yields by diluting bicelle solutions composed of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and DHPC (1,2-dihexanoyl-sn-glycero-3-phosphocholine). After passing through the microfluidic chip, DMPC bilayer domain coalesced and formed vesicle, while DHPC could dissolve into an aqueous phase. The membrane properties of the vesicles exhibited a highly ordered phase, indicating that DHPCs were removed from transitioned vesicles after dilution in a microfluidic chip. Moreover, by controlling the dilution ratio, vesicles of various sizes ranging from 90 to 480 nm with an enhanced monodispersity can be obtained without any additional process.
An NMR Study on the Conformation of Substance P in Acidic Bicelles
Baek, Seung-Bin,Lim, Sung-Chul,Lee, Hyeong-Ju,Lee, Hee-Cheon,Kim, Chul Korean Chemical Society 2011 Bulletin of the Korean Chemical Society Vol.32 No.10
The conformation of a neuropeptide, substance P (SP), in isotropic (q = 0.5) acidic bicelles was investigated using two-dimensional NMR techniques. By the nuclear Overhauser effect (NOE) cross peaks between SP and long-chain lipid molecules SP was probed to bind on the flat surface of the disc-like bicelles. Structural analysis of NMR data indicated that the helical conformation of SP extended to the C-terminal region of Leu10 as well as in the mid-region from Pro4 to Phe8. As compared with the conformations of SP bound on the sodium dodecylsulfate (SDS) or the dodecylphosphocholine (DPC) micelles with curved surfaces, the surface curvature of the membrane mimics was found to be one of the major factors inducing the biologically relevant conformation of SP. The negative surface charge of the membrane is also a key factor inducing both the binding of SP on the membrane and its biologically active structure.